The present invention relates to micro-chamber plate, and particularly to an analytic micro-chamber plate in which multiple reaction solutions containing a primer or a probe selectively reacted with each nucleic acid are reacted without cross contamination in order to analyze a biological sample solution containing multiple nucleic acids, thereby measuring and analyzing a fluorescence value in real time.
Further, the present invention related to a method of manufacturing the analytic micro-chamber plate.
Further, the present invention relates to a method of manufacturing a micro-chamber plate with a built-in sample, which is used in the manufacturing of the analytic micro-chamber plate.
Further, the present invention relates to an apparatus set for manufacturing the sample containing micro-chamber plate.
Generally, a micro-chamber is a container which is formed of silicon wafer, glass, metal, plastic or the like and in which a fine reaction less than a few micro-liters. The micro-chamber plate is a plate in which the micro-chambers are arranged in two dimensions and of which one side surface is formed to be sealed after a sample is injected therethrough.
Meanwhile, there has been developed a real-time PCR (Polymerase Chain Reaction) method which can measure a fluorescence value increasing in proportion to an amount of genes in real-time, while performing a PCR.
In the real-time PCR method, while the PCR is carried out, the fluorescence value generated from a product of the PCR is measured in every cycle, and the cycle when the fluorescence value larger than a desired value is generated is checked, thereby quantitatively analyzing an initial concentration of a specific gene in a sample.
In the real-time PCR method, there are some advantages in that an electrophoresis process following the PCR is not needed, and it is possible to decide a concentration of a gene having a specific base sequence in the range of 109 or more (“A-Z of Quantitative PCR” edited by Stephen A. Bustin 2004-2006 International University, “Real-time PCR” edited by M. Tevfik Dorak 2006 Taylor & Francis Group).
There had been proposed various kinds of real-time PCR apparatuses for performing the real-time PCR method. For example, there is a conventional real-time PCR apparatus which can analyze 96 or 384 genes using a standard 96-well or 394-well plate, thereby analyzing a plurality of samples (Light cycler 480 manufactured by Roche, ABI 7500, 7900).
In the conventional real-time PCR apparatus manufactured by Roche, in which a reaction sample of 10˜500 μl is used, however, there is a problem that it is not possible to analyze a large number of genes compared to a large amount of used sample.
In order to solve the problem, various methods which can simultaneously analyze multiple samples in a shorter time by reducing a used amount of the reaction sample using a MEMS (Micro Electro Mechanical Systems) technology have been proposed, and thus a method using a micro-chamber array plate has been also proposed.
The method using a micro-chamber array plate includes a step of injecting a reaction solution into a micro-chamber, a step of sealing the reaction solution in each micro-chamber, and a reacting and analyzing step. In a method of separately applying a sample solution in each micro-chamber, a transparent micro-chamber plate for cell culture is covered by a semi-permeable membrane so as to individually isolate the micro-chambers, and one cell is cultivated in each micro-chamber, and a Taqman reaction solution is supplied after removing a culture medium, and the micro-chamber is sealed by transparent oil, and then a fluorescence value is measured at a bottom surface of the plate (YASUDA, Kenji EP 1,541,678 A1, JP 2002245900 NUCLEIC ACID ANALYSIS CHIP AND NUCLEIC ACID ANALYZER).
In the above-mentioned method, however, since different solutions have to be applied to each micro-chamber using a pipette, much time is spent on that. Particularly, an auto-pipetting system is needed in order to inject the sample in 1,536 or more micro-chambers. Herein, in order to apply the different solutions, it takes lots of time due to a cleaning process which has to be performed before applying each of the different solutions. Thus, there is a problem in that it is difficult to use the 384 plates or more.
Secondly, in order to solve the problem, there had been proposed a reactor by E. Tamiya, Hidenori Nagai et al., in which a micro-chamber is formed by treating a silicon wafer in a photolithography process and a chemical etching process (Anal. Chem. 2001 73, 1043-1047, Development of a Micro-chamber Array for Picoliter PCR).
In the reactor, a micro-slide cover glass is used to prevent the evaporation of a PCR solution. However, since cross contamination of the PCR solution is occurred when covering or separating the cover glass, it is inconvenient that the cover glass has to be removed while a water-repellent film is interposed between the cover glass and the wafer, the water-repellent film has to be removed after drying the PCR solution and then an analysis process has to be performed. Further, there is a problem in that it cannot be used in qPCR technology.
Thirdly, in order to solve the problem of using in the qPCR technology, there had been developed another micro-chamber array by Y. Matsubara et al. belonging to the same laboratory, in which a primer is applied to a micro-chamber formed on a wafer using a micro-array device and then dried (7th International Conference on Miniaturized Chemical and Biochemical Analysis Systems Oct. 5-9, 2003, Squaw Valley Calif. USA).
The micro-chamber array uses a method in which mineral oil is applied on a chip so as to completely cover the micro-chambers, and then a PCR solution is dripped on the mineral oil of the reactor using a nano-jet pipetting system.
In the method, 1,248 micro-chamber array chips having a volume of 50 nano-liters (0.65×0.65×0.2 mm) are manufactured by treating a 1 inch×3 inch silicon wafer in a photolithography process and a chemical etching process, a primer and a Taqman probe solution are dripped in the micro-chambers using the nano jet pipetting system and dried, and then the mineral oil is coated thereon so that each micro-chamber is isolated and sealed.
In case of the micro-chamber array manufactured by the third method, since a mixed solution of a Taq DNA polymerase and a sample DNA is injected on the mineral oil using the nano jet pipetting system so as to be dripped in each micro-chamber, there is an advantage in that it is possible to successfully carry out the PCR in the micro-chambers without cross contamination of each reaction component.
However, in this method, there are some problems that a separate nano jet pipetting system for micro-array is needed to inject the solution, it takes lots of time to perform the pipetting operation and there is also a high risk of the cross contamination among the reaction solutions due to flowing of the mineral oil when the plate is moved. Further, in a temperature cycling reaction, bubbles are generated at high temperature. Meanwhile, the aqueous solution in each micro-chamber is formed into a globular shape due to a hydrophobic effect between the oil and the aqueous solution, thereby causing a lens effect. Thus since excitation light and luminescence is scattered and dispersed upon the optical measurement, the measurement error is increased.
Fourthly, there had been also developed a picotiter plate in which micro-chambers are formed by the photolithography process and the chemical etching process like in the third method but a lot more reactions than in the third method can be performed (John H. Leamon et al., A massively parallel PocoTiterPlate based platform for discrete pico-liter-scale polymerase chanin reactions, Electrophoresis 2003, 24, 3769-3777).
In the fourth method, it is possible to independently carry out 300,000 PCRs with an amount of 39.5 pl. However, since a carrier in which primers/probes are immobilized is needed, it cannot be applied to a real-time quantitative PCR method in which uniform optical characteristics are required.
Fifthly, in U.S. Pat. No. 5,948,637, there has been proposed a reactor called “a film reactor (or a DNA card)” for reacting a small amount of sample.
The film reactor is form of a three-layered very thin film. Detailedly, a lower film forms a lower surface of the reactor, a middle film forms a side surface of the reactor and an upper film forms an injection hole. After a small amount of sample solution is injected into the film reactor by using a pipette, the injection hole has to be completely sealed. If the injection hole is not completely sealed, there is a problem that the reaction solution is evaporated upon the PCR. Further, since the film reactor has a complicated structure in order to treat a few thousands of samples, it is substantially impossible to manufacture it.
Sixthly, in WO 02/40158 and U.S. Pat. No. 6,232,114, there is disclosed a reaction plate which can carry out 1,535 fluorescence analysis reactions with a standard ELISA plate scale.
In the sixth method, multiple through-holes are formed in the plate, and a transparent film having a small fluorescence amount is fused so as to form a plurality of reaction vessels. After the sample is received in each reaction vessel, the reaction vessels are sealed with the transparent film and the reaction is carried out. Upper and lower surfaces of the reaction plate are formed to be transparent, and excitation light is applied through one side surface, and then the fluorescence is measured through the other side surface.
In the sixth method, however, different primer and probe have to be respectively injected into each micro-chamber in order to analyze a great number of genes. In case of a plate for analyzing a great number of samples, since a few thousands of different solutions are injected at the micro-chambers, a special pipetting system such as a nano-liter pipetting system is needed, much time is spent on that and also erroneous injections may be occurred. Further, since the micro-chamber cannot be completely filled with the solution, bubbles are generated, and the water vapors are formed at an upper portion of the micro-chamber when raising the temperature, and thus the optical measurement is disturbed by the scattering.
Seventhly, in PCT/KR2008/005635 invented by the invention of the present application, there is disclosed a reaction plate using a micro-chamber plate that a porous membrane for injecting a sample is formed at one side surface thereof and an optical measuring part is formed at the other side surface thereof.
In the seventh method, multiple through-holes are formed in the plate, and a transparent film having a small fluorescence amount is fused at one side surface thereof so as to form a plurality of reaction vessels. After the sample is received in each reaction vessel, the other side surface thereof is sealed with the porous membrane through which the sample solution can be injected, and the reaction is carried out. In the reaction plate, the sample solution is injected through the porous membrane, and mineral oil is sealingly dripped on the injection surface, and then excitation light is applied and the fluorescence is measured through the optical measuring part formed at the other side surface thereof.
However, in the seventh method, since the injection part and the optical measuring part are formed separately, it has a complicated structure. And the oil layer formed on the injection part becomes transparent, and thus a deviation problem in the measurement results may be occurred according to a stained state of a bottom surface. Further, the injection part on which the mineral oil is dripped may be directed downward in order to perform the reaction and measurement. At this time, the mineral oil having a relatively lower density than the sample may be introduced into the micro-chamber, and thus the scattering may be occurred.
Eighthly, in PCT/KR2008/005635, there is disclosed “The micro-chamber plate, manufacturing method thereof”.
However, since the eighth method has a structure that a sample to be injected is directly applied to a porous membrane, it has some problems as follows: 1) in case that the injection of the sample is achieved by a vacuum, centrifugal force is applied in order to prevent running-out of the sample while the vacuum is applied. Herein, discharging of gas through pores of the porous membrane is disturbed by the centrifugal force and surface tension of the sample; 2) since the gas in the micro-chamber is compressed by the centrifugal force and thus a volume thereof is contracted, the gas does not obtain enough buoyancy to get out of the micro-chamber through the membrane, but is remained in the form of small bubbles and then expanded again in the measurement condition of atmospheric pressure, thereby disturbing the measurement.
Therefore, a new micro-chamber plate is needed, in which the sample can be easily injected into the plurality of micro-chambers, the cross-contamination is not occurred, light generated from the sample can be precisely measured in real time without possibility that the optical measuring part is contaminated with the sample or the like.